A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Clients' experiences on North America's first take-home injectable opioid agonist treatment (iOAT) program: a qualitative study.

BACKGROUND: To support public health measures during the COVID-19 pandemic, oral opioid agonist treatment (OAT) take-home doses were expanded in Western countries with positive results. Injectable OAT (iOAT) take-home doses were previously not an eligible option, and were made available for the first time in several sites to align with public health measures. Building upon these temporary risk-mitigating guidelines, a clinic in Vancouver, BC continued to offer two of a possible three daily doses of take-home injectable medications to eligible clients. The present study explores the processes through which take-home iOAT doses impacted clients' quality of life and continuity of care in real-life settings. METHODS: Three rounds of semi-structured qualitative interviews were conducted over a period of seventeen months beginning in July 2021 with eleven participants receiving iOAT take-home doses at a community clinic in Vancouver, British Columbia. Interviews followed a topic guide that evolved iteratively in response to emerging lines of inquiry. Interviews were recorded, transcribed, and then coded using NVivo 1.6 using an interpretive description approach. RESULTS: Participants reported that take-home doses granted them the freedom away from the clinic to have daily routines, form plans, and enjoy unstructured time. Participants appreciated the greater privacy, accessibility, and ability to engage in paid work. Furthermore, participants enjoyed greater autonomy to manage their medication and level of engagement with the clinic. These factors contributed to greater quality of life and continuity of care. Participants shared that their dose was too essential to divert and that they felt safe transporting and administering their medication off-site. In the future, all participants would like more accessible treatment such as access longer take-home prescriptions (e.g., one week), the ability to pick-up at different and convenient locations (e.g., community pharmacies), and a medication delivery service. CONCLUSIONS: Reducing the number of daily onsite injections from two or three to only one revealed the diversity of rich and nuanced needs that added flexibility and accessibility in iOAT can meet. Actions such as licencing diverse opioid medications/formulations, medication pick-up at community pharmacies, and a community of practice that supports clinical decisions are necessary to increase take-home iOAT accessibility.

The development of highly potent and selective small molecule correctors of Z α1-antitrypsin misfolding.

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors.

Differential gene expression analysis reveals pathways important in early post-traumatic osteoarthritis in an equine model.

BACKGROUND: Post-traumatic osteoarthritis (PTOA) is a common and significant problem in equine athletes. It is a disease of the entire joint, with the synovium thought to be a key player in disease onset and progression due to its role in inflammation. The development of effective tools for early diagnosis and treatment of PTOA remains an elusive goal. Altered gene expression represents the earliest discernable disease-related change, and can provide valuable information about disease pathogenesis and identify potential therapeutic targets. However, there is limited work examining global gene expression changes in early disease. In this study, we quantified gene expression changes in the synovium of osteoarthritis-affected joints using an equine metacarpophalangeal joint (MCPJ) chip model of early PTOA. Synovial samples were collected arthroscopically from the MCPJ of 11 adult horses before (preOA) and after (OA) surgical induction of osteoarthritis and from sham-operated joints. After sequencing synovial RNA, Salmon was used to quasi-map reads and quantify transcript abundances. Differential expression analysis with the limma-trend method used a fold-change cutoff of log2(1.1). Functional annotation was performed with PANTHER at FDR < 0.05. Pathway and network analyses were performed in Reactome and STRING, respectively. RESULTS: RNA was sequenced from 28 samples (6 preOA, 11 OA, 11 sham). "Sham" and "preOA" were not different and were grouped. Three hundred ninety-seven genes were upregulated and 365 downregulated in OA synovium compared to unaffected. Gene ontology (GO) terms related to extracellular matrix (ECM) organization, angiogenesis, and cell signaling were overrepresented. There were 17 enriched pathways, involved in ECM turnover, protein metabolism, and growth factor signaling. Network analysis revealed clusters of differentially expressed genes involved in ECM organization, endothelial regulation, and cellular metabolism. CONCLUSIONS: Enriched pathways and overrepresented GO terms reflected a state of high metabolic activity and tissue turnover in OA-affected tissue, suggesting that the synovium may retain the capacity to support healing and homeostasis in early disease. Limitations of this study include small sample size and capture of one point post-injury. Differentially expressed genes within key pathways may represent potential diagnostic markers or therapeutic targets for PTOA. Mechanistic validation of these findings is an important next step.

Photosynthetic Responses of Turf-forming Red Macroalgae to High CO2 Conditions.

Seaweeds are important components of near-shore ecosystems as primary producers, foundation species, and biogeochemical engineers. Seaweed communities are likely to alter under predicted climate change scenarios. We tested the physiological responses of three perennial, turf-building, intertidal rhodophytes, Mastocarpus stellatus, Osmundea pinnatifida, and the calcified Ellisolandia elongata, to elevated pCO2 over 6 weeks. Responses varied between these three species. E. elongata was strongly affected by high pCO2 , whereas non-calcified species were not. Elevated pCO2 did not induce consistent responses of photosynthesis and respiration across these three species. While baseline photophysiology differed significantly between species, we found few clear effects of elevated pCO2 on this aspect of macroalgal physiology. We found effects of within-species variation in elevated pCO2 response in M. stellatus, but not in the other species. Overall, our data confirm the sensitivity of calcified macroalgae to elevated pCO2 , but we found no evidence suggesting that elevated pCO2 conditions will have a strong positive or negative impact on photosynthetic parameters in non-calcified macroalgae.

Tolerance of Impatiens balsamina L., and Crotalaria retusa L. to grow on soil contaminated by used lubricating oil: A comparative study.

Screening of plant species with an ability to grow on contaminated soil is the most critical step in the planning of a phytoremediation program. While flourishing growth of Impatiens balsamina L. and Crotalaria retusa L. has been observed in areas adjacent to automobile service stations in Sri Lanka, no systematic study of their tolerance to used lubricating oil (ULO) contaminated soil has been carried out. Therefore, the aim of the present study was to investigate the comparative responses of I. balsamina L. and C. retusa L. to soil contaminated with ULO. Both species exhibited 100% seed germination in soils treated with 1%-5% w/w ULO. After 120 h exposure, root lengths and biomass of germinated seedlings of both species were significantly (p < 0.05) reduced in all treatments above 3% w/w ULO. The measured growth parameters of plants following 90 d exposure to 0.5-3% w/w ULO, indicated significant (p < 0.05) negative effects on I. balsamina and C. retusa at >1% w/w and >2% w/w ULO, respectively. There were no significant effects on chlorophyll content or root anatomy of either species under any treatments. Therefore, we concluded that I. balsamina can tolerate up to 1% of ULO and C. retusa up to 2% w/w ULO without displaying any negative effects. Comparatively higher biodegradation of ULO in the rhizosphere, root nodule formation, increases in root length and root hair density are all possible strategies for the exhibited higher tolerance of C. retusa. Therefore, the overall results indicate that C. retusa has the greater potential to be used in phytoremediation of ULO contaminated soils. The findings of the present study will be beneficial in planning phytoremediation program for ULO contaminated soil.

Synthesis and Conformational Analysis of Fluorinated Uridine Analogues Provide Insight into a Neighbouring-Group Participation Mechanism.

Fluorinated nucleoside analogues have attracted much attention as anticancer and antiviral agents and as probes for enzymatic function. However, the lack of direct synthetic methods, especially for 2',3'-dideoxy-2',3'-difluoro nucleosides, hamper their practical utility. In order to design more efficient synthetic methods, a better understanding of the conformation and mechanism of formation of these molecules is important. Herein, we report the synthesis and conformational analysis of a 2',3'-dideoxy-2',3'-difluoro and a 2'-deoxy-2'-fluoro uridine derivative and provide an insight into the reaction mechanism. We suggest that the transformation most likely diverges from the SN1 or SN2 pathway, but instead operates via a neighbouring-group participation mechanism.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms.

Following the physiological complementary/parallel Celis-Plá et al., by inhibiting extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and cytokinin specific binding protein (p38), we assessed the role of the mitogen-activated protein kinases (MAPK) pathway in detoxification responses mediated by chronic copper (10 µM) in U. compressa. Parameters were taken at 6, 24, and 48 h, and 6 days (d). H2O2 and lipid peroxidation under copper and inhibition of ERK, JNK, or p38 alone increased but recovered by the sixth day. By blocking two or more MAPKs under copper, H2O2 and lipid peroxidation decayed even below controls. Inhibition of more than one MAPK (at 6 d) caused a decrease in total glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and ascorbate (reduced ascorbate (ASC) + dehydroascorbate (DHA)), although in the latter it did not occur when the whole MAPK was blocked. Catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRX) ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione synthase (GS), were downregulated when blocking more than one MAPK pathway. When one MAPK pathway was blocked under copper, a recovery and even enhancement of detoxification mechanisms was observed, likely due to crosstalk within the MAPKs and/or other signalling processes. In contrast, when more than one MAPK pathway were blocked under copper, impairment of detoxification defences occurred, demonstrating that MAPKs were key signalling mechanisms for detoxification in macroalgae.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion/Extrusion Mechanisms and Photosynthesis.

There is currently no information regarding the role that whole mitogen activated protein kinase (MAPK) pathways play in counteracting environmental stress in photosynthetic organisms. To address this gap, we exposed Ulva compressa to chronic levels of copper (10 µM) specific inhibitors of Extracellular Signal Regulated Kinases (ERK), c-Jun N-terminal Kinases (JNK), and Cytokinin Specific Binding Protein (p38) MAPKs alone or in combination. Intracellular copper accumulation and photosynthetic activity (in vivo chlorophyll a fluorescence) were measured after 6 h, 24 h, 48 h, and 6 days of exposure. By day 6, when one (except JNK) or more of the MAPK pathways were inhibited under copper stress, there was a decrease in copper accumulation compared with algae exposed to copper alone. When at least two MAPKs were blocked, there was a decrease in photosynthetic activity expressed in lower productivity (ETRmax), efficiency (αETR), and saturation of irradiance (EkETR), accompanied by higher non-photochemical quenching (NPQmax), compared to both the control and copper-only treatments. In terms of accumulation, once the MAPK pathways were partially or completely blocked under copper, there was crosstalk between these and other signaling mechanisms to enhance metal extrusion/exclusion from cells. Crosstalk occurred among MAPK pathways to maintain photosynthesis homeostasis, demonstrating the importance of the signaling pathways for physiological performance. This study is complemented by a parallel/complementary article Rodríguez-Rojas et al. on the role of MAPKs in copper-detoxification.

Pac13 is a Small, Monomeric Dehydratase that Mediates the Formation of the 3'-Deoxy Nucleoside of Pacidamycins.

The uridyl peptide antibiotics (UPAs), of which pacidamycin is a member, have a clinically unexploited mode of action and an unusual assembly. Perhaps the most striking feature of these molecules is the biosynthetically unique 3'-deoxyuridine that they share. This moiety is generated by an unusual, small and monomeric dehydratase, Pac13, which catalyses the dehydration of uridine-5'-aldehyde. Here we report the structural characterisation of Pac13 with a series of ligands, and gain insight into the enzyme's mechanism demonstrating that H42 is critical to the enzyme's activity and that the reaction is likely to proceed via an E1cB mechanism. The resemblance of the 3'-deoxy pacidamycin moiety with the synthetic anti-retrovirals, presents a potential opportunity for the utilisation of Pac13 in the biocatalytic generation of antiviral compounds.

The sensitivity of an hydroponic lettuce root elongation bioassay to metals, phenol and wastewaters.

The root elongation bioassay is one of the most straightforward test methods used for environmental monitoring in terms of simplicity, rapidity and economy since it merely requires filter paper, distilled water and Petri dishes. However, filter paper as a support material is known to be problematic as it can reduce the sensitivity of the test. The newly developed hydroponic method reported here differs from the conventional root elongation method (US EPA filter paper method) in that no support material is used and the exposure time is shorter (48 h in this test versus 120 h in the US EPA test). For metals, the hydroponic test method was 3.3 (for Hg) to 57 (for Cu) times more sensitive than the US EPA method with the rank orders of sensitivity, estimated from EC50 values, being Cu≥Cd>Ni≥Zn≥Hg for the former and Hg≥Cu≥Ni≥Cd≥Zn for the latter methods. For phenol, the results did not differ significantly; EC50 values were 124 mg L(-1) and 108-180 mg L(-1) for the hydroponic and filter paper methods, respectively. Lettuce was less sensitive than daphnids to wastewaters, but the root elongation response appears to be wastewater-specific and is especially sensitive for detecting the presence of fluorine. The new hydroponic test thus provides many practical advantages, especially in terms of cost and time-effectiveness requiring only a well plate, a small volume of distilled water and short exposure period; furthermore, no specialist expertise is required. The method is simpler than the conventional EPA technique in not using filter paper which can influence the sensitivity of the test. Additionally, plant seeds have a long shelf-life and require little or no maintenance.

MAGNETIC RESONANCE IMAGING SCORING OF AN EXPERIMENTAL MODEL OF POST-TRAUMATIC OSTEOARTHRITIS IN THE EQUINE CARPUS.

Magnetic resonance imaging (MRI) is the most sensitive imaging modality to detect the early changes of osteoarthritis. Currently, there is no quantifiable method to tract these pathological changes over time in the horse. The objective of this experimental study was to characterize the progression of MRI changes in an equine model of post-traumatic osteoarthritis using a semiquantitative scoring system for whole-organ evaluation of the middle carpal joint. On day 0, an osteochondral fragment was created in one middle carpal joint (OCI) and the contralateral joint (CON) was sham-operated in 10 horses. On day 14, study horses resumed exercise on a high-speed treadmill until the completion of the study (day 98). High-field MRI examinations were performed on days 0 (preosteochondral fragmentation), 14, and 98 and scored by three blinded observers using consensus agreement. Images were scored based on 15 independent articular features, and scores were compared between and within-groups. On days 14 and 98, OCI joints had significantly (P ≤ 0.05) higher whole-organ median scores (29.0 and 31.5, respectively), compared to CON joints (21.5 and 20.0, respectively). On day 14, OCI joints showed significant increases in high-signal bone lesion scores, and osteochondral fragment number and size. On day 98, high-signal bone lesion, low-signal bone lesion, osteophyte formation, cartilage signal abnormality, subchondral bone irregularity, joint effusion, and synovial thickening scores were significantly increased in OCI joints. Study results suggest that the MRI whole-organ scoring system reported here may be used to identify onset and progression of pathological changes following osteochondral injury.

Inter-population comparisons of copper resistance and accumulation in the red seaweed, Gracilariopsis longissima.

Copper (Cu) resistance and accumulation of five populations of the red seaweed Gracilariopsis longissima collected from sites in south west England (Fal Estuary, Helford Estuary and Chesil Fleet) that differ in their degree of Cu contamination was assessed under controlled laboratory conditions, on two separate occasions (April and October). The effects of a range of Cu concentrations (0-250 µg l(-1)) on relative growth rates was the same for all populations with reductions observable at concentrations as low as 12 µg l(-1) and cessation of growth at 250 µg l(-1). There was no significant difference in the calculated EC(50) values for the April and October samples, with means of 31.1 and 25.8 µg l(-1), respectively. Over the range of concentrations used in this study, copper content increased linearly and the pattern of accumulation was the same for all populations at both time periods. From the linear regressions of the pooled data a concentration factor of 2.25 × 10(3) was calculated. These results imply that G. longissima has an innate tolerance to Cu and that populations have not evolved copper-tolerant ecotypes. In laboratory studies, accumulated Cu was released when transferred to 'clean' seawater with approximately 80% being lost after 8 days, with no significant difference between populations in their response. The results from a 30 days in situ transplantation experiment using two populations from the Fal Estuary provided further evidence for dynamic changes in Cu content in response to changes in Cu bioavailability. The findings in this study are discussed in the context of implications for seaweed biomonitoring.

Interactions of silver nanoparticles with the marine macroalga, Ulva lactuca.

The marine macroalga, Ulva lactuca, has been exposed for 48 h to different concentrations of Ag added as either silver nanoparticles (AgNP) or aqueous metal (AgNO(3)) and the resulting toxicity, estimated from reductions in quenching of chlorophyll-a fluorescence, and accumulation of Ag measured. Aqueous Ag was toxic at available concentrations as low as about 2.5 µg l(-1) and exhibited considerable accumulation that could be defined by the Langmuir equation. AgNP were not phytotoxic to the macroalga at available Ag concentrations up to at least 15 µg l(-1) and metal measured in U. lactuca was attributed to a physical association of nanoparticles at the algal surface. At higher AgNP concentrations, a dose-response relationship was observed that was similar to that for aqueous Ag recorded at much lower concentrations. These findings suggest that AgNP are only indirectly toxic to marine algae through the dissolution of Ag(+) ions into bulk sea water, albeit at concentrations orders of magnitude greater than those predicted in the environment.

Commercial Potential of the Cyanobacterium Arthrospira maxima: Physiological and Biochemical Traits and the Purification of Phycocyanin.

Arthrospira maxima is a natural source of fine chemicals for multiple biotechnological applications. We determined the optimal environmental conditions for A. maxima by measuring its relative growth rate (RGR), pigment yield, and photosynthetic performance under different pH and temperature conditions. RGR was highest at pH 7-9 and 30 °C. Chlorophyll a, phycocyanin, maximal quantum yield (Fv/Fm), relative maximal electron transport rate (rETRmax), and effective quantum yield (ΦPSII) were highest at pH 7-8 and 25 °C. Interestingly, phycoerythrin and allophycocyanin content was highest at 15 °C, which may be the lowest optimum temperature reported for phycobiliprotein production in the Arthrospira species. A threestep purification of phycocyanin (PC) by ultrafiltration, ion-exchange chromatography, and gel filtration resulted in a 97.6% purity of PC.

Toxicity of the amphoteric surfactant, cocamidopropyl betaine, to the marine macroalga, Ulva lactuca.

The degradation of the synthetic, amphoteric surfactant, cocamidopropyl betaine (CAPB) and its toxicity to the marine macroalga, Ulva lactuca, has been evaluated using several different physiological test end-points over different periods of exposure up to 120 h. Droplet surface angle measurements revealed that, following a period of acclimation of about 24 h, CAPB began to degrade and that primary degradation was complete within 120 h. Effective quantum yield (∆F/F(m)') and relative growth rates (RGRs) were the most sensitive measures of phytotoxicity, with CAPB concentrations at and above 10 mg l(-1) eliciting irreversible, time-dependent and/or dose-dependent responses. Cell membrane damage, estimated from measurements of ion leakage, was detected only at a concentration of 40 mg l(-1) after 48 h of exposure to CAPB but by 120 h damage was evident at all measured concentrations above 10 mg l(-1). These observations suggest that both CAPB and its metabolites are intrinsically toxic to U. lactuca. The findings of this study are discussed in terms of the environmental consequences of applying CAPB to control harmful algal blooms.

Sensitivity of spore germination and germ tube elongation of Saccharina japonica to metal exposure.

The sensitivity of early life stages of the brown seaweed Saccharina japonica to six metals (Cd, Cu, Hg, Ni, Pb, Zn) and two waste-water samples were investigated and a new toxicity bioassay developed. The two endpoints used were spore germination and germ tube elongation with an exposure time of 24 h. Optimal test conditions determined for photon irradiance, pH, salinity and temperature were darkness, pH 8, 35‰ and 15°C, respectively. The toxicity ranking of five metals was: Hg (EC(50) of 41 and 42 µg l(-1)) > Cu (120 and 81 µg l(-1)) > Ni (2,009 and 1,360 µg l(-1)) > Zn (3,024 and 3,897 µg l(-1)) > Pb (4,760 and 4,429 µg l(-1)) > Cd (15,052 and 7,541 µg l(-1)) for germination and germ tube elongation, respectively. The sensitivities to Cd, Cu and Ni were greater in germ tube elongation than in germination process. When tested against two different waste-water samples (processed animal and printed circuit board waste-water) values of EC(50) were between 21.29 and 32.02% for germination and between 5.33 and 8.98% for germ tube elongation. Despite differences in their chemical composition, the toxic effects of waste-water samples, as indicated by EC(50) values, did not differ significantly for the same endpoints. The CV range for both germination and germ tube elongation was between 4.61 and 37.69%, indicating high levels of precision of the tests. The results compare favourably with those from more established test procedures employing micro- and macroalgae. The advantages and potential limitations of the bioassay for the assessment of anthropogenic impacts on coastal ecosystems and commercial cultivation areas in near-shore environments are discussed.

Surgical resolution of an avulsion fracture of the peroneus tertius origin in a giraffe (Giraffa camelopardalis reticulata).

A 4-mo-old, 185-kg male giraffe (Giraffa camelopardalis reticulata) was presented due to stifle effusion and lameness of 3-wk duration. Radiographs revealed a fracture of the extensor fossa of the lateral trochlear ridge of the femur at the origin of the peroneus tertius. Under anesthesia, dysfunction of the reciprocal apparatus was documented by flexing the stifle while the tarsus remained extended. An avulsion fracture of the origin of the peroneus tertius and extensor digitorum longus muscle was diagnosed. An exploratory arthroscopy of the femorotibial joint was followed by arthrotomy to excise the large bone fragment from its soft tissue attachments. Because of the fractious temperament of the animal, postoperative care was restricted to stall rest for 3 mo, and no postoperative complications arose. Only a mild residual lameness remained by 6 mo after surgery.